Sambrook J. Molecular cloning: a laboratory manual. Vol.3 / J.Sambrook, D.W.Russell. - 3rd ed. - New York: Cold Spring Harbor Laboratory, 2001. - xxvii, P.15.1-18.136 (various pagings): ill. - Incl. bibl. ref. and index. - ISBN 978-087969577-4  Место хранения:   040 | Институт биофизики СО РАН | Красноярск | Библиотека

Оглавление / Contents

Preface ....................................................... xxi Acknowledgments ............................................. xxiii Cold Spring Harbor Protocols Online ........................... xxv Quotation Credits ........................................... xxvii Volume 3 Chapter 15 Expression of Cloned Genes in Escherichia coli ............... 15.1 INTRODUCTION PROTOCOLS 1 Expression of Cloned Genes in E. coli Using IPTG- inducible Promoters ...................................... 15.14 2 Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter ............................ 15.20 3 Expression of Cloned Genes in E. coli Using the Bacteriophage λ p1 Promoter .......................... 15.25 4 Expression of Secreted Foreign Proteins Using the Alkaline Phosphatase Promoter (phoA) and Signal Sequence ................................................. 15.30 ·Additional Protocol: Subcellular Localization of PhoA Fusion Proteins ......................................... 15.35 5 'Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose .................... 15.36 6 Purification of Maltose-binding Fusion Proteins by Affinity Chromatography on Amylose Resin ................. 15.40 7 Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography ............... 15.44 ·Alternative Protocol: Elution of Polyhistidine-tagged Proteins from Metal Affinity Columns Using Decreasing pH ...................................................... 15.47 ·Additional Protocol: Regeneration of NTA-Ni2+ - Agarose ................................................. 15.48 8 Purification oi Expressed Proteins from Inclusion Bodies ................................................... 15.49 ·Additional Protocol: Refolding Solubilized Proteins Recovered from Inclusion Bodies ......................... 15.53 INFORMATION PANELS Expression of Cloned Genes .................................. 15.55 E. coli Expression Systems .................................. 15.56 LacZ Fusions ................................................ 15.57 Chaotropic Agents ........................................... 15.60 Chapter 16 Introducing Cloned Genes into Cultured Mammalian Cells ....... 16.1 INTRODUCTION PROTOCOLS 1 DNA Transfection Mediated by Lipofection .................. 16.7 ·Additional Protocol: Histochemical Staining of Cell Monolayers for β-Gaiactosidase ......................... 16.13 2 Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs .................................. 16.14 ·Alternative Protocol: High-efficiency Calcium- phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs ....................................... 16.19 3 Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA ........................ 16.21 ·Alternative Protocol: Calcium-phosphate-mediated Transfection of Adherent Cells .......................... 16.25 ·Alternative Protocol: Calcium-phosphate-mediated Transfection of Cells Growing in Suspension ............. 16.26 4 Transfection Mediated by DEAE-Dextran: High-efficiency Method ................................................... 16.27 ·Alternative Protocol: Transfection Mediated by DEAE- Dextran: Increased Cell Viability ....................... 16.32 5 DNA Transfection by Electroporation ...................... 16.33 6 DNA Transfection by Biolistics ........................... 16.37 ·Additional Protocol: Histochemical Staining of Cell Monolayers or Tissue for β-Glucuronidase ............... 16.42 7 DNA Transfection Using Polybrene ......................... 16.43 INFORMATION PANELS Cotransformation ............................................ 16.47 Selective Agents for Stable Transformation .................. 16.48 Lipofection ................................................. 16.50 Transfection of Mammalian Cells with Calcium Phosphate-DNA Coprecipitates ........................................... 16.52 Chloroquine Diphosphate ..................................... 16.53 Electroporation ............................................. 16.54 Chapter 17 Analysis of Gene Expression in Cultured Mammalian Cells ...... 17.1 INTRODUCTION PROTOCOLS CIs-acting Regions and Trans-acting Factors 1 Mapping Protein-binding Sites on DNA by DNase I Footprinting .............................................. 17.4 ·Alternative Protocol: Mapping Protein-binding Sites on DNA by Hydroxyl Radical Footprinting .................... 17.12 2 Gel Retardation Assays for DNA-binding Proteins .......... 17.13 ·Additional Protocol: Supershift Assays .................. 17.17 ·Additional Protocol: Competition Assays ................. 17.17 3 Mapping DNase-l-hypersensitive Sites ..................... 17.18 Analysis of Primary Transcripts 4 Transcriptional Run-on Assays ............................ 17.23 Reporter Assays Introduction to Reporter Assays: CAT, Luciferase, and β-galactosidase (Protocols 5-7) ......................... 17.30 5 Measurement of Chloramphenicol Acetyltransferase in Extracts of Mammalian Cells Using Thin-layer Chromatography ........................................... 17.33 ·Alternative Protocol: Measurement of CAT by Extraction with Organic Solvents ................................... 17.40 ·Alternative Protocol: Measurement of CAT following Diffusion of Reaction Products into Scintillation Fluid ................................................... 17.41 6 Assay for Luciferase in Extracts of Mammalian Cells ...... 17.42 ·Alternative Protocol: Using a Scintillation Counter to Measure Luciferase ................................... 17.46 ·Alternative Protocol: Assay for Luciferase in Cells Growing in 96-well Plates ............................... 17.47 7 Assay for β-galactosidase in Extracts of Mammalian Cells .................................................... 17.48 Inducible Systems 8 Tetracycline as Regulator of Inducible Gene Expression in Mammalian Cells ....................................... 17.52 Stage 1: Stable Transfection of Fibroblasts with pTet-tTAk ....................................... 17.60 Stage 2: Stable Transfection of Inducible tTA- expressing NIH-3T3 Cells with Tetracycline- regulated Target Genes .......................... 17.65 Stage 3: Analysis of Protein Expression in Transfected Cells ........................................... 17.68 ·Alternative Protocol: Tetracycline-regulated Induction of Gene Expression in Transiently Transfected Cells Using the Autoregulatory tTA System ..................... 17.70 9 Ecdysone as Regulator of Inducible Gene Expression in Mammalian Cells .......................................... 17.71 INFORMATION PANELS Footprinting DNA ............................................ 17.75 Gel Retardation Assays ...................................... 17.78 Baculoviruses and Baculovirus Expression Systems ............ 17.81 Green Fluorescent Proteins .................................. 17.84 Epitope Tagging ............................................. 17.90 Chloramphenicol Acetyltransferase ........................... 17.94 Luciferase .................................................. 17.96 β-galactosidase ............................................. 17.97 Chapter 18 Protein Interaction Technologies ............................. 18.1 INTRODUCTION PROTOCOLS 1 Two-hybrid and Other Two-component Systems ................ 18.6 Stage 1: Characterization of a Bait-LexA Fusion Protein ......................................... 18.17 ·Alternative Protocol: Assay of β-galactosidase Activity by Chloroform Overlay 18.28 Stage 2: Selecting an Interactor .............................................. 18.30 Stage 3: Second Confirmation of Positive Interactions .... 18.38 ·Alternative Protocol: Rapid Screen for Interaction Trap Positives ............................................... 18.46 2 Detection of Protein-Protein Interactions Using Far Western with GST Fusion Proteins ......................... 18.48 ·Additional Protocol: Refolding of Membrane-bound Proteins ................................................ 18.53 ·Alternative Protocol: Detection of Protein-Protein Interactions with Anti-GST Antibodies ................... 18.54 3 Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique ........................ 18.55 4 Identification of Associated Proteins by Coimmunoprecipitation .................................... 18.60 5 Probing Protein Interactions Using GFP and Fluorescence Resonance Energy Transfer ................................ 18.69 Stage 1: Labeling Proteins with Fluorescent Dyes ......... 18.80 Stage 2: Cell Preparation for FLIM-FRET Analysis ......... 18.84 Alternative Protocol: Preparation of Fixed Cells for ·FLIM-FRET Analysis ...................................... 18.87 ·Alternative Protocol: Microinjection of Live Cells ...... 18.88 Stage 3: FLIM-FRET Measurements .......................... 18.90 Analysis of Interacting Proteins with Surface Plasmon Resonance Spectroscopy Using BIAcore ..................... 18.96 Stage 1: Preparation of the Capture Surface and Test Binding ........................................ 18.104 Stage 2: Kinetic Analysis of the Antibody-Antigen Interaction .................................... 18.108 INFORMATION PANELS Filamentous Phage Display .................................. 18.115 Genomics and the Interaction Trap .......................... 18.123 Interaction Trap and Related Technologies .................. 18.125 Appendices 1 Preparation of Reagents and Buffers Used in Molecular Cloning ................................................... A1.1 2 Media ..................................................... A2.1 3 Vectors and Bacterial Strains ............................. A3.1 4 Enzymes Used in Molecular Cloning ......................... A4.1 5 Inhibitors of Enzymes ..................................... A5.1 6 Nucleic Acids ............................................. A6.1 7 Codons and Amino Acids .................................... A7.1 8 Commonly Used Techniques in Molecular Cloning ............. A8.1 9 Detection Systems ......................................... A9.1 10 DNA Array Technology ..................................... A10.1 11 Bioinformatics ........................................... A11.1 12 Cautions ................................................. A12.1 13 Suppliers ................................................ A13.1 14 Trademarks ............................................... A14.1 Appendix References ............................................ R1 INDEX, 1.1 ..................................................... R1